CTNNBL1 facilitates the association of CWC15 with CDC5L and is required to maintain the abundance of the Prp19 spliceosomal complex

In order to catalyse the splicing of messenger RNA, multiple proteins and RNA components associate and dissociate in a dynamic highly choreographed process. The Prp19 complex is a conserved essential part of the splicing machinery thought to facilitate the conformational changes the spliceosome undergoes during catalysis. Dynamic protein interactions often involve highly disordered regions that are difficult to study by structural methods. Using amine crosslinking and hydrogen–deuterium exchange coupled to mass spectrometry, we describe the architecture of the Prp19 sub-complex that contains CTNNBL1. Deficiency in CTNNBL1 leads to delayed initiation of cell division and embryonic lethality. Here we show that in vitro CTNNBL1 enhances the association of CWC15 and CDC5L, both core Prp19 complex proteins and identify an overlap in the region of CDC5L that binds either CTNNBL1 or CWC15 suggesting the two proteins might exchange places in the complex. Furthermore, in vivo, CTNNBL1 is required to maintain normal levels of the Prp19 complex and to facilitate the interaction of CWC15 with CDC5L. Our results identify a chaperone function for CTNNBL1 within the essential Prp19 complex, a function required to maintain the integrity of the complex and to support efficient splicing.

[1]  G. Hong,et al.  Nucleic Acids Research , 2015, Nucleic Acids Research.

[2]  J. Ellenberg,et al.  SNW1 enables sister chromatid cohesion by mediating the splicing of sororin and APC2 pre‐mRNAs , 2014, The EMBO journal.

[3]  K. Gould,et al.  Structural and Functional Insights into the N-Terminus of Schizosaccharomyces pombe Cdc5 , 2014, Biochemistry.

[4]  M. Howell,et al.  Functional genomics identifies a requirement of pre-mRNA splicing factors for sister chromatid cohesion , 2014, The EMBO journal.

[5]  K. Nagai,et al.  Structural studies of the spliceosome: zooming into the heart of the machine☆ , 2014, Current opinion in structural biology.

[6]  Eun-Jung Kim,et al.  Structural insights into the novel ARM-repeat protein CTNNBL1 and its association with the hPrp19-CDC5L complex. , 2014, Acta crystallographica. Section D, Biological crystallography.

[7]  N. Rhind,et al.  Endogenous U2·U5·U6 snRNA complexes in S. pombe are intron lariat spliceosomes , 2014, RNA.

[8]  L. Zou,et al.  PRP19 transforms into a sensor of RPA-ssDNA after DNA damage and drives ATR activation via a ubiquitin-mediated circuitry. , 2014, Molecular cell.

[9]  H. Urlaub,et al.  Mass spectrometry–based relative quantification of proteins in precatalytic and catalytically active spliceosomes by metabolic labeling (SILAC), chemical labeling (iTRAQ), and label-free spectral count , 2014, RNA.

[10]  Jun Huang,et al.  The PSO4 Protein Complex Associates with Replication Protein A (RPA) and Modulates the Activation of Ataxia Telangiectasia-mutated and Rad3-related (ATR)* , 2014, The Journal of Biological Chemistry.

[11]  Christopher M Johnson,et al.  Structural and mutational analysis reveals that CTNNBL1 binds NLSs in a manner distinct from that of its closest armadillo-relative, karyopherin α , 2013, FEBS letters.

[12]  K. Sträßer,et al.  Splicing and beyond: the many faces of the Prp19 complex. , 2013, Biochimica et biophysica acta.

[13]  C. Will,et al.  The Prp19 Complex Directly Functions in Mitotic Spindle Assembly , 2013, PloS one.

[14]  Andrew N. Holding,et al.  Hekate: Software Suite for the Mass Spectrometric Analysis and Three-Dimensional Visualization of Cross-Linked Protein Samples , 2013, Journal of proteome research.

[15]  Z. Du,et al.  The structure of full-length human CTNNBL1 reveals a distinct member of the armadillo-repeat protein family. , 2013, Acta crystallographica. Section D, Biological crystallography.

[16]  M. Neuberger,et al.  Deficiency in spliceosome-associated factor CTNNBL1 does not affect ongoing cell cycling but delays exit from quiescence and results in embryonic lethality in mice , 2013, Cell cycle.

[17]  Roger L. Williams,et al.  Dynamics of the Phosphoinositide 3-Kinase p110δ Interaction with p85α and Membranes Reveals Aspects of Regulation Distinct from p110α , 2011, Structure.

[18]  M. Seizl,et al.  The Prp19 Complex Is a Novel Transcription Elongation Factor Required for Trex Occupancy at Transcribed Genes Functional Analysis of the Rna Polymerase Ii C-terminal Domain Kinase Ctk1 in the Yeast , 2022 .

[19]  Henning Urlaub,et al.  Semiquantitative Proteomic Analysis of the Human Spliceosome via a Novel Two-Dimensional Gel Electrophoresis Method , 2011, Molecular and Cellular Biology.

[20]  J. Manley,et al.  The RNA polymerase II C-terminal domain promotes splicing activation through recruitment of a U2AF65-Prp19 complex. , 2011, Genes & development.

[21]  M. Neuberger,et al.  CTNNBL1 Is a Novel Nuclear Localization Sequence-binding Protein That Recognizes RNA-splicing Factors CDC5L and Prp31 , 2011, The Journal of Biological Chemistry.

[22]  Henning Urlaub,et al.  Characterization of purified human Bact spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis. , 2010, RNA.

[23]  R. O’Keefe,et al.  The function of the NineTeen Complex (NTC) in regulating spliceosome conformations and fidelity during pre-mRNA splicing. , 2010, Biochemical Society transactions.

[24]  Dongsup Kim,et al.  Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe , 2010, Nature Biotechnology.

[25]  Henning Urlaub,et al.  Determination of protein stoichiometry within protein complexes using absolute quantification and multiple reaction monitoring. , 2010, Analytical chemistry.

[26]  Henning Urlaub,et al.  Molecular Architecture of the Human Prp19/CDC5L Complex , 2010, Molecular and Cellular Biology.

[27]  Nianxiang Zhang,et al.  Cdc5L interacts with ATR and is required for the S‐phase cell‐cycle checkpoint , 2009, EMBO reports.

[28]  J. Brüning,et al.  PLRG1 Is an Essential Regulator of Cell Proliferation and Apoptosis during Vertebrate Development and Tissue Homeostasis , 2009, Molecular and Cellular Biology.

[29]  C. Will,et al.  The Spliceosome: Design Principles of a Dynamic RNP Machine , 2009, Cell.

[30]  M. Neuberger,et al.  Interaction between antibody-diversification enzyme AID and spliceosome-associated factor CTNNBL1. , 2008, Molecular cell.

[31]  C. Guthrie,et al.  Modifications target spliceosome dynamics , 2008, Nature Structural &Molecular Biology.

[32]  J. Qin,et al.  Isolation of XAB2 Complex Involved in Pre-mRNA Splicing, Transcription, and Transcription-coupled Repair* , 2008, Journal of Biological Chemistry.

[33]  Henning Urlaub,et al.  Protein Composition and Electron Microscopy Structure of Affinity-Purified Human Spliceosomal B Complexes Isolated under Physiological Conditions , 2006, Molecular and Cellular Biology.

[34]  Zoran Obradovic,et al.  Length-dependent prediction of protein intrinsic disorder , 2006, BMC Bioinformatics.

[35]  H. Katinger,et al.  SNEV is an evolutionarily conserved splicing factor whose oligomerization is necessary for spliceosome assembly , 2005, Nucleic acids research.

[36]  Peter Tompa,et al.  The role of structural disorder in the function of RNA and protein chaperones , 2004, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[37]  M. Wilm,et al.  A subset of human 35S U5 proteins, including Prp19, function prior to catalytic step 1 of splicing , 2004, The EMBO journal.

[38]  M. Konarska,et al.  Suppression of multiple substrate mutations by spliceosomal prp8 alleles suggests functional correlations with ribosomal ambiguity mutants. , 2004, Molecular cell.

[39]  T. Kitamura,et al.  Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics. , 2003, Experimental hematology.

[40]  W. Tsai,et al.  The Prp19p-Associated Complex in Spliceosome Activation , 2003, Science.

[41]  K. Gould,et al.  Structural insights into the U-box, a domain associated with multi-ubiquitination , 2003, Nature Structural Biology.

[42]  J. Kollár,et al.  Sequence, gene structure, and expression pattern of CTNNBL1, a minor-class intron-containing gene--evidence for a role in apoptosis. , 2003, Genomics.

[43]  F. Rösel,et al.  Amplification of the BCAS2 gene at chromosome 1p13.3-21 in human primary breast cancer. , 2002, Cancer letters.

[44]  K. Gould,et al.  Proteomics Analysis Reveals Stable Multiprotein Complexes in Both Fission and Budding Yeasts Containing Myb-Related Cdc5p/Cef1p, Novel Pre-mRNA Splicing Factors, and snRNAs , 2002, Molecular and Cellular Biology.

[45]  A. Lamond,et al.  A Direct Interaction between the Carboxyl-terminal Region of CDC5L and the WD40 Domain of PLRG1 Is Essential for Pre-mRNA Splicing* , 2001, The Journal of Biological Chemistry.

[46]  B. Kuster,et al.  Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry , 2000, The EMBO journal.

[47]  E. J. Vicente,et al.  PSO4: a novel gene involved in error-prone repair in Saccharomyces cerevisiae. , 1989, Mutation research.

[48]  P. Romero,et al.  Sequence complexity of disordered protein , 2001, Proteins.

[49]  PROTEINS: Structure, Function, and Bioinformatics Suppl 7:176–182 (2005) Exploiting Heterogeneous Sequence Properties Improves Prediction of Protein Disorder , 2022 .